| To obtain a wild macro-fungi which have bleaching and antibacterial function, optimize its bleaching conditions and the fermentation parameters of producing antimicrobial substances and research the physicochemical properties of the active ingredients and its separation and purification process. Taking 53 strains of wild macro-fungi as studying object, The active strains were screened in terms of the methods of absorbance, well diffusion assay and hypha growth rate, which were identified by ITS-5.8 rDNA sequences analysis and its bleaching conditions and the fermentation parameters of producing antimicrobial substances were optimized by response surface analysis and orthogonal test.9 strains with decolorization effects on Malachite Green dye were screened from 53 species of wild macro-fungi, of which 0331 strain was the strongest. its optimal fermentation medium was composed of potato 20%, corn juice 2%, KH2PO4 0.3%, MgSO4·7H2O 0.15%. The optimal decolorization conditions were of 100 mg/L initial concentrations of dye,6 mL of fermented liquid content,6 mmol/L of Mg2+concentration, 6.0 of pH,50 ℃ of temperature, and 150 r/min of oscillating speed of shaker. Under such conditions, the decolorization rate reached 92.6% within 1h. Fe2+ had a strong inhibitory effect on decolorization.12 strains with antimicrobial activity were screened, of which strains 0612-9 and 0331 exhibited stronger and more broad-spectrum antimicrobial activity, especially the strain 0612-9. The optimal medium for 0331 was composed of (in g/L): potatoes 181.94, wheat bran 3.00, Sucrose 37.628, KH2PO4 4.00, MgSO4·7H2O 1.00, VB1 0.0018. The optimal culture conditions were as follows:the initial pH value of medium 5.0, the fermentation period 10 d, packing volume 110 mL/250 mL, inoculation 8 mL/100 mL, rotate speed 180 r/min, temperature 28 ℃, respectively. The optimal fermentation parameters for strain 0612-9 were potatoes 270.53 g/L, wheat bran 7.429 g/L, glucose 30 g/L, MgSO4·7H2O 0.4551 g/L, KH2PO4 2 g/L, The initial pH value of medium was 4.0, the fermentation period of 11 d, packing volume 100 mL/250 mL, inoculation 5 mL/100 mL, rotate speed 180 r/min, temperature 27 ℃ respectively. The strain 0612-9 was cultured in a volume of 15 liter fermentation tank and the fermentation kinetics were studied. The reducing sugar and amino nitrogen of fermentation liquid were up after a decline, overall the value of DO and pH keep decline, the biomass and the titer were decline after up. The extracellular antibacterial substances produced by the strain 0612-9 could remain active less than 60 ℃, pH 1.0-6.0, under ultraviolet irradiation, and had similar to polarity of ethyl acetate. It could be deduced preliminary by TLC that the active compounds could contain steroid three terpene, terpenoids, phenols, coumarin and so on. The antimicrobial part of Ethyl acetate extracts from fermented liquor of strain 0612-9 was isolated followed by 4 silica gel column of elution variety and more pure active fractions were obtained. The active fractions were further separation by HPLC and the compounds with antimicrobial activity were obtained. The optimal elution program for HPLC were as below:injection volume of 100 μL, the mobile phase is 20%-55% acetonitrile, velocity 1.5 mL/min, wavelength of detection was 207 nm. Analysis of the ITS sequence preliminarily suggested that the strain 0331 belongs to the Cerrena sp. and the strain 0612-9 belongs to the Gymnopus sp.Decolorization effects on malachite green dye of zymotic fluid produced by Cerrena sp. were reported for the first time. Gymnopus sp. can be used as a novel source of antimicrobial agents. |
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